Inactivation of Escherichia coli O157:H7 on inoculated alfalfa seeds with ozonated water and heat treatment

Alfalfa seeds inoculated with a five-strain mixture of Escherichia coli O157:H7 were immersed in water containing 4, 8, 10, and 21 ppm of ozone for 2, 4, 8, 16, 32, and 64 min at 4degreesC. Direct ozone sparging of seeds in water was used as an alternative mode of ozone treatment. Ozone-sparged seeds were also subsequently exposed to heat treatment at 40, 50, and 60degreesC for 3 h. Populations of E. coli O157:H7 on untreated and treated seeds were determined by spread plating diluted samples on tryptic soy agar supplemented with 50 mug/ml of nalidixic acid. Since E. coli O157:H7 was released from inoculated seeds during treatment with ozone, the rate of release of cells from inoculated seeds soaked in 0.1% peptone water for up to 64 min was also determined. The overall reduction of E. coli O157:H7 on seeds treated with ozonated water without continuous sparging ranged from 0.40 to 1.75 log(10) CFU/g (59.6 to 98.2%), whereas reductions for control seeds were 0.32 to 1.03 log(10) CFU/g (51.7 to 90.5%). Treatment with higher ozone concentrations enhanced inactivation, but contact time longer than 8 min did not result in significantly higher reductions (P > 0.05). For seeds treated by ozone sparging, a 1.12-log(10) CFU/g (92.1%) reduction was achieved using a 2-min contact time, and a 2.21-log(10) CFU/g (99.4%) reduction was achieved with a 64-min contact time. The corresponding reductions for control seeds were 0.71 log(10) CFU/g (79.5%) and 2.21 log(10) CFU/g (99.4%), respectively, Treatment of ozone-sparged seeds at 60degreesC for 3 h reduced the population to an undetectable level by direct plating (4 to 4.8 log(10) CFU/g), although survivors were detected by enrichment. Ozone did not have a detrimental effect on seed germination percentage.